SUMMARY There is a fundamental gap in our understanding of how mutations in enzymes of the base excision repair (BER) pathway may affect a protein's function, global conformation and its interactions with protein partners, and how these changes can lead to initiation of carcinogenesis. A powerful combination of structural, biochemical and cellular methods will be employed to study the molecular mechanisms of the human BER glycosylases that repair oxidative damage. These enzymes are the ?first responders? as their task is to recognize and excise oxidized bases in DNA while leaving normal bases untouched. The central hypothesis of this program project is that defects in BER proteins can drive human carcinogenesis and affect responses to cancer treatments. The objective of Project 2 is to understand, at the biochemical and structural levels, how the BER glycosylases recognize and process oxidized lesions, how the flexible regions of the proteins influence activity and interactions with DNA or protein partners, and how single-point mutations affect the protein form and function and may ultimately initiate carcinogenesis. Guided by strong preliminary data the three aims of this proposal will 1- determine the biochemical and molecular mechanisms of lesion recognition by the NEIL glycosylases, 2- elucidate the molecular mechanisms of inhibition, activation and dimerization of NTHL1 glycosylase, and 3- evaluate the effects of BER glycosylase mutations by determining the biochemical and structural characteristics of these variants and assessing their biological phenotypes. These aims will use biochemical and structural biology methods, such as X-ray crystallography and small angle X-ray scattering (SAXS), which will be used to determine the shape and form of the full-length glycosylases and potential changes brought upon by mutations. The structure/function studies from Project 2 will work synergistically with the phenotypical characterization in human cells carried out by Project 1. Our work also dovetails with the work done in Project 3 on NTHL1 and substrate hand off, and the single-molecule studies carried out by Project 4. Core A will provide bioinformatics and statistical support for the study of the human variants. Purified proteins and human cell cultures will be provided by Core B. We anticipate that this work will provide fundamental insights into the molecular mechanisms of BER glycosylases. These results are expected to have a positive impact because they will reveal how amino acid substitutions in DNA glycosylases lead to initiation of carcinogenesis, knowledge that will be beneficial for predicting cancer susceptibility and optimizing treatment strategies.